Modulation by LL-37 of the Responses of Salivary Glands to Purinergic Agonists

Abstract

The interaction of mice submandibular gland cells with LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), a cationic peptide with immunomodulatory properties, was investigated. LL-37 at a concentration that did not affect the integrity of the cells increased the uptake of calcium and activated a calciuminsensitive phospholipase A 2 (PLA 2 ). The small release of ATP induced by LL-37 could not account for this stimulation because apyrase did not significantly block the response to LL-37. The divalent cation magnesium inhibited the response to LL-37, but this inhibition was probably nonspecific because it also inhibited the in vitro bacteriostatic effect of the peptide. The increase of calcium uptake by LL-37 was not affected by, a rather specific inhibitor of P2X 7 receptors in mice. LL-37 also increased [Ca 2ϩ ] i in cells from mice invalidated for these receptors. LL-37 had no effect on the response to carbachol. It inhibited the increase of [Ca 2ϩ ] i and the activation of phospholipase D by ATP. It potentiated the activation of the PLA 2 by the nucleotide. Finally, LL-37 increased the fluidity of the plasma membrane of submandibular gland cells. In conclusion, our results suggest that LL-37 is an autocrine regulator of submandibular gland cells. It does not stimulate mouse P2X 7 receptors but modulates their responses.Cathelicidins are proteins involved in the first phases of our defenses against pathogens. Their isolation relied on the analogy of their N-terminal proregions with cathelin, an inhibitor of cathepsin L originally isolated from bovine leukocytes (Ritonja et al., 1989). This highly conserved proregion (approximately 100 amino acids) shares many similarities with cystatins, inhibitors of the cysteine proteases. The Cterminal domain of cathelicidins varies among species both in terms of length (12-100 amino acids) and structure. The N-and C-terminal domains are separated by a sequence recognized by proteases. The digestion in the extracellular medium of the propeptides by elastase (Panyutich et al., 1997) or proteinase 3 (Sorensen et al., 2001) releases the C-terminal peptides originally described as antimicrobial but that now prove to be more immunomodulatory than antimicrobial in physiological conditions (Bowdish et al., 2005). Most of the studies on cathelicidins have focused on the C-terminal peptides. These peptides are ubiquitous. They are secreted by macrophages, lymphocytes, epithelial cells, keratinocytes, cells lining the upper respiratory tree, and vaginal cells (Bals and Wilson, 2003). LL-37, the only peptide from human origin, is derived from an antibacterial protein of 18 kDa. LL-37 and its only analog in mouse, the cathelinrelated antimicrobial peptide, are cationic and transform from a random coil in aqueous solution to an amphipathic ␣-helix at the contact of a membrane (Yeaman and Yount, 2003). The peptide binds to the bacteria by electrostatic interactions between the positive charges on one side of its

Involvement of the P2X₇ Purinergic Receptor and c-Jun N-Terminal and Extracellular Signal-Regulated Kinases in Cyclooxygenase-2 and Prostaglandin E₂ Induction by LL-37

“…Similarly to IL-1β-induced PGE 2 formation, which is mediated by enhanced COX-2 gene expression in gingival fibroblasts [15], PGE 2 levels were also raised by LL-37 via the specificity of COX-2 activation, consistent with our previous result showing that elevated PGE 2 levels by hBD-3 treatment result from induced COX-2 expression [17]. The inducible effect of LL-37 on COX-2 expression and PGE 2 production in HGFs is relevant to the increased activity of phospholipase A 2 , an upstream molecule involved in arachidonic acid metabolism, induced by LL-37 in mouse submandibular acinar cells [26]. …”

“…In addition, LL-37 has been reported to be directly chemotactic for human neutrophils, monocytes, and T cells through formyl peptide receptor-like 1, a G i protein-coupled receptor [36], and to activate keratinocyte migration via transactivation of the epidermal growth factor receptor by phosphorylation of ERK MAPK [37]. LL-37 has also been found to induce IL-8 production through phosphorylation of ERK1/2 via the P2X 7 receptor in HGFs [21], submandibular gland cells [26], and human embryonic kidney 293 cells [38]. Accordingly, it is possible that LL-37 may either interact with its cognate receptors or internalize into the cells, or both, to upregulate COX-2 expression and PGE 2 production in HGFs.…”

A parasitic helminth-derived peptide that targets the macrophage lysosome is a novel therapeutic option for autoimmune disease

“…Both the human LL-37 and the murine CRAMP (cathelicidin-related antimicrobial peptide, an orthologue of human LL-37) have been shown in vitro to reduce the activation of IL-1␤ by the Nlrp3 inflammasome in macrophages (Pochet et al, 2006;Seil et al, 2010;Hu et al, 2014). In all of these cases, ATP activated the inflammasome protein complex and inhibition was achieved via interaction of the cathelicidin peptide with the receptor for ATP, P2X7.…”

Purinergic receptors in the endocrine and exocrine pancreas

“…The microbial peptide LL37, from the family of cathelicidins, could also activate P2X 7 receptors [117]. Although LL37 is found in salivary glands [118] and its role in P2X 7 receptor stimulation has been discussed [119,120], there are no reports of cathelicidins in the pancreas. It has also been proposed that extracellular Cl − could regulate the P2X 7 receptor in parotid gland ducts [119,121].…”